3/19/2009

TORTORA FUNKE CASE

ninth edition

MICROBIOLOGY
an introduction

Microbial Growth
Microbial growth is the increase in number of cells,
not cell size

6
Microbial
Growth
PowerPoint LectureSlidePresentationpreparedbyChristineL.Case
Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

The Requirements for Growth:

Physical Requirements

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Temperature

Temperature
Minimum growth temperature
Optimum growth temperature
Maximum g
growth temperature
p

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Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Psychrotrophs

Psychrotrophs

Figure 6.1

Grow between 0C and 20-30C

Cause food spoilage

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Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Figure 6.2

3/19/2009

The Requirements for Growth:

Physical Requirements

The Requirements for Growth:

Physical Requirements

pH

Osmotic pressure

Most bacteria grow between pH 6.5 and 7.5

Hypertonic environments, increase salt or sugar,

Molds and yeasts grow between pH 5 and 6

cause plasmolysis

Acidophiles
p
g
grow in acidic environments

Extreme or obligate
g
halophiles
p
require
q
high
g osmotic
pressure
Facultative halophiles tolerate high osmotic pressure

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

The Requirements for Growth:

Physical Requirements

The Requirements for Growth:

Chemical Requirements
Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs
p use CO2

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The Requirements for Growth:

Chemical Requirements

Figure 6.4

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The Requirements for Growth:

Chemical Requirements

Nitrogen
In amino acids and proteins

Trace elements

Most bacteria decompose proteins

Inorganic elements required in small amounts

Some bacteria use NH4+ or NO3

Usually as enzyme cofactors

A few bacteria use N2 in nitrogen fixation

Sulfur
S lf
In amino acids, thiamine and biotin
Most bacteria decompose proteins
Some bacteria use SO42 or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43 is a source of phosphorus
Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

3/19/2009

The Requirements for Growth:

Chemical Requirements

Toxic Forms of Oxygen

Oxygen (O2)

Singlet oxygen: O2 boosted to a higher-energy state

Superoxide free radicals: O2

Peroxide anion: O22

Hydroxyl radical (OH)

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Table 6.1

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The Requirements for Growth:

Chemical Requirements

Culture Media

Organic growth factors

Culture medium: Nutrients prepared for microbial

Organic compounds obtained from the environment

Vitamins, amino acids, purines, and pyrimidines

growth
Sterile: No living microbes
Inoculum: Introduction of microbes into medium
Culture: Microbes growing in/on culture medium

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Agar

Culture Media

Complex polysaccharide

Chemically defined media: Exact chemical composition

Used as solidifying agent for culture media in Petri

plates, slants, and deeps
Generallyy not metabolized by
y microbes

is known
Complex media: Extracts and digests of yeasts, meat,
or p
plants

Liquefies at 100C

Nutrient broth

Solidifies ~40C

Nutrient agar

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Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

3/19/2009

Culture Media

Growth in Continuous Culture

A continuous culture is an open system in which
fresh media is continuously added to the culture at
a constant rate, and old broth is removed at the
same rate.
This method is accomplished in a device called a
chemostat.
Typically, the concentration of cells will reach an
equilibrium level that remains constant as long as
the nutrient feed is maintained.

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Tables 6.2, 6.4

Basic Chemostat System

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Our Chemostat System

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Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Anaerobic Culture Methods

Anaerobic Culture Methods

Reducing media

Anaerobic

Contain chemicals (thioglycollate or oxyrase) that

jar

combine O2
Heated to drive off O2

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Figure 6.5

3/19/2009

Anaerobic Culture Methods

Capnophiles Require High CO2

Anaerobic

Candle jar

chamber

CO2-packet

Figure 6.6

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Figure 6.7

Selective Media

Selective Media

Suppress unwanted

Inhibits the growth of some bacteria while selecting for

microbes and

the growth of others

Example:

encourage desired

Brilliant Green Agar

g

microbes.

dyes inhibit the growth of Gram (+) bacteria

selects for Gram (-) bacteria
Most G.I. Tract infections are caused by Gram (-)
bacteria
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Figure 6.9bc

Selective Media
EMB (Eosin Methylene Blue)
dyes inhibit Gram (+) bacteria

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Differential Media
Make it easy to distinguish colonies of different
microbes.

selects for Gram (-) bacteria

G.I. Tract infections caused by
y Gram (-)
()
bacteria

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Figure 6.9a

3/19/2009

Some media are both selective and

differential

Selective and Differential Media

Mannitol salt agar is both selective and differential

Selective:

Staphylococcus aereus can grow on mannitol salt agar that has a

high concentration of salt; the growth of other organisms will be
inhibited

Differential:

Staphylococcus aureus ferments mannitol and the medium will

change color
Other organisms that grow on high salt will grow on mannitol salt
agar but may not ferment mannitol; the media will not change
colors

Mannitol Salt Agar

used to identify Staphylococcus aureus
Mannitol
M
i l Salt
S l A
Agar
High salt conc. (7.5%) inhibits most bacteria
sugar Mannitol
pH Indicator (Turns Yellow when acid)

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Selective and Differential Media

Enrichment Media
Encourages growth of desired microbe

MacConkeys Agar
used to identify Salmonella
MacConkeys Agar
Bile salts and crystal violet (inhibits Gram (+)
bacteria)
lactose
pH Indicator
Many Gram (-) enteric non-pathogenic bacteria can
ferment lactose, Salmonella can not
Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Assume a soil sample contains a few phenol-degrading

bacteria and thousands of other bacteria
Inoculate phenol-containing culture medium with the
soil and incubate
Transfer 1 ml to another flask of the phenol medium
and incubate
Transfer 1 ml to another flask of the phenol medium
and incubate
Only phenol-metabolizing bacteria will be growing
Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Streak Plate
A pure culture contains only one species or strain.
A colony is a population of cells arising from a single
cell or spore or from a group of attached cells.
A colonyy is often called a colony-forming
y
g unit ((CFU).
)

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Figure 6.10ab

3/19/2009

Preserving Bacteria Cultures

Reproduction in Prokaryotes

Deep-freezing: 50to 95C

Binary fission

Lyophilization (freeze-drying): Frozen (54 to 72C)

Budding
Conidiospores (actinomycetes)

and dehydrated in a vacuum

Fragmentation
g
of filaments

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Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Binary Fission

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Figure 6.11

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Figure 6.12b

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Figure 6.13

If 100 cells growing for 5 hours produced 1,720,320

cells:

PLAY

Animation: Bacterial Growth

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3/19/2009

1. Lag Phase
Bacteria are first introduced into an environment or
media
Bacteria are checking out their surroundings
cells are veryy active metabolicallyy
# of cells changes very little
1 hour to several days

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Figure 6.14

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

3. Stationary Phase

2. Log Phase
Rapid cell growth (exponential growth)

Death rate = rate of reproduction

population doubles every generation

cells begin to encounter environmental stress

lack of nutrients

microbes are sensitive to adverse conditions

antibiotics

lack of water

anti-microbial agents

not enough space

metabolic wastes
oxygen
pH

Endospores would form now

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

4. Death Phase

Measuring Microbial Growth

Death rate > rate of reproduction

Direct methods

Indirect methods

Due to limiting factors in the environment

Plate counts

Turbidity

Filtration

Metabolic activity

MPN

Dryy weight
g

Direct microscopic count

Dry weight

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

3/19/2009

Direct Measurements of Microbial Growth

Plate Count

Plate counts: Perform serial dilutions of a sample

Inoculate Petri
plates from serial
dilutions

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Figure 6.15, step 1

Plate Count

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Figure 6.16

Direct Measurements of Microbial Growth

After incubation, count colonies on plates that have

Filtration

25-250 colonies (CFUs)

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Figure 6.15

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Figure 6.17

Direct Measurements of Microbial Growth

Direct Measurements of Microbial Growth

Multiple tube

Direct microscopic count

MPN test.
Count positive
tubes and
compare to
statistical
MPN table.

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Figure 6.18b

Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings

3/19/2009

Direct Measurements of Microbial Growth

Estimating Bacterial Numbers

by Indirect Methods
Turbidity

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Figure 6.19, steps 1, 3

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Figure 6.20

10